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Biocatalysts and methods for hydroxylation of compounds

A technology of compounds and substrates, applied in the field of biocatalysts

Active Publication Date: 2017-09-08
CODEXIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although recombinant whole cells expressing cloned proline hydroxylases are more suitable for large-scale industrial production processes, the use of whole cells limits reaction conditions, such as high substrate concentration variations; constraining the substrates that can be used with whole cells Types are those that are permeable to cells; and lead to undesired by-products that must be separated from the final product

Method used

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  • Biocatalysts and methods for hydroxylation of compounds
  • Biocatalysts and methods for hydroxylation of compounds
  • Biocatalysts and methods for hydroxylation of compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0462] Example 1: Synthesis, optimization and screening of engineered proline hydroxylase polypeptides

[0463] Gene Synthesis and Optimization : The polynucleotide sequence encoding the reported wild-type cis-4-proline hydroxylase polypeptide from S. meliloti as represented by SEQ ID NO:2 was synthesized as the gene of SEQ ID NO:1. The synthetic gene of SEQ ID NO: 1 was cloned into the pCK110900 vector system (see eg, US Patent Application Publication 20060195947, which is hereby incorporated by reference) and subsequently expressed in E. coli W3110fhuA. E. coli W3110 expresses a proline hydroxylase polypeptide under the control of the lac promoter. In silico simulation of enzyme structure based on sequence alignment with other proline hydroxylases and docked substrate proline, with active site, peptide loop, solution / substrate interface, and potential stability Positionally related residue positions were identified and subjected to mutagenesis. These first round variants...

Embodiment 2

[0464] Example 2: Production of engineered proline hydroxylase

[0465] Engineered proline hydroxylase polypeptides were produced in E. coli W3110 under the control of the lac promoter. Enzyme preparations for HTP, DSP, and SFP assays were prepared as follows.

[0466] High-throughput (HTP) growth, expression, and lysate preparation . Cells were picked and grown overnight at 30°C, 200 rpm, 85% humidity in LB medium containing 1% glucose and 30 μg / ml chloramphenicol (CAM). A 20 μl aliquot of the overnight growth was transferred to a deep well plate containing 380 μl of 2xTB growth medium containing 30 μg / ml CAM, 1 mM IPTG, and incubated at 30°C, 200 rpm, 85% humidity for ~18h. The cell culture was centrifuged at 4000 rpm, 4°C for 10 min, and the medium was discarded. The cell pellet was resuspended in 100 μl lysis buffer (50 mM phosphate buffer, pH 6.3, containing 100 μM molar salts (i.e., (NH 4 ) 2 Fe(SO 4 ) 2 ), 0.5mg / mL PMBS (polymyxin B sulfate) and 1mg / mL lysozyme...

Embodiment 3

[0469] Example 3: Analysis procedure

[0470] Method 1-HPLC Analysis of HTP Determination Reaction: In a 96 deep well format assay block, 10 μL of the reaction solution was diluted with 230 μL of 5% sodium bicarbonate solution followed by 160 μL of dansyl chloride solution (6 mg / mL dansyl chloride in MeCN). Plates were heat sealed, centrifuged, and placed in an incubator at 55°C for 45 minutes. When the derivatization of dansyl chloride was completed, the reaction solution changed from yellow to light yellow. Where the solution remained yellow, the plate was heated for another 15 min. After incubation, the plates were centrifuged at 4000 rpm for 5 min. A 200 [mu]L aliquot of the supernatant was transferred to a 96 Corning plate for HPLC analysis. The final concentration of substrate was less than 0.25 g / L.

[0471] The quenched reaction was subjected to HPLC analysis under the following conditions.

[0472]

[0473] Conversion of compound (2) to compound (1) was dete...

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Abstract

The present disclosure provides engineered proline hydroxylase polypeptides for producing hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, hosts capable of expressing the engineered proline hydroxylases Cells, and methods of using the engineered proline hydroxylases to prepare compounds useful in producing active pharmaceutical agents. The present disclosure provides engineered proline hydroxylase biocatalysts, polynucleotides encoding the biocatalysts, methods of making them, and methods of using these engineered biocatalysts to make hydroxylated compounds.

Description

technical field [0001] The present disclosure relates to biocatalysts for the hydroxylation of compounds. [0002] References to Sequence Listings, Tables or Computer Programs [0003] The official text of the sequence listing is submitted as an ASCII format text file and instructions via EFS-Web at the same time, the file name is "CX2-095WO2_ST25.txt", the creation date is May 2, 2013, and the size is 433,542 bytes. The Sequence Listing submitted via EFS-Web is part of this specification and is hereby incorporated by reference in its entirety. Background technique [0004] Because of the constrained conformation of proline, proline derivatives with functional groups on the carbocycle are useful building blocks for the synthesis of pharmaceutical compounds. One such derivative, hydroxylated proline, is a starting material for the synthesis of various therapeutic compounds including carbapenem antibiotics (see, e.g., Altamura et al., 1995, J Med Chem .38(21):4244-56), angi...

Claims

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Application Information

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IPC IPC(8): C12N9/04
CPCC12N9/0071C12P13/24C12P17/12C12P17/188C12Y114/11002C12P13/04Y02P20/52
Inventor 陈海滨荣贵·彭法比安·卡比罗尔阿努潘·苟呵尔涛·李杰弗里·C·穆尔玛蒂娜·金塔纳尔-欧德罗宏·杨史蒂文·J·科利尔德里克·史密斯
Owner CODEXIS INC
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